Methanol-assimilating propagation of microbial cells

ABSTRACT

A strain belonging to the new species Pseudomonas methanolica is aerobically cultured in a nutrient medium containing methanol as a substrate and the propagated microbial cells are recovered from the culture liquor.

United States Patent [1 1 Terui et al.

METHANOL-ASSIMILATING PROPAGATION OF MICROBIAL CELLS Inventors: Gyozo Terui; .Nobuo Takada;

Hidekazu Sawada, all of Osaka,

Japan Assignee: Gyozo Terui, Osaka, Japan Filed: Nov. 10, 1971 Appl. No.: 197,437

Foreign Application Priority Data Dec. 11, 1970 Japan 45/110957 US. Cl 195/49, 195/115, 195/96,

. 195/140 Int. Cl Cl2b l/00 Field of Search 195/96, 28 R, 1,

[ 51 Aug. 28, 1973 Primary Examiner-Alvin E. Tanenholtz Assistant ExaminerThomas G. Wiseman Attorney-Joseph M. Fitzpatrick, Nels T. Lippert et al.

[57] ABSTRACT A strain belonging to the new species Pseudomonas methanolica is aerobically cultured in a nutrient medium containing methanol as a substrate and the propagated microbial cells are recovered from the culture liquor.

5 Claims, 4 Drawing Figures Patented Aug. 28, 1973 2 Sheets-Sheet 2 k wJJ W a g 0 41 n n n u o on u 0 Huh w u l- 1 C .m m. n w M 1 3 m 9 S E o h F 1 4 m m w 5 2\ v cozm cwucou Al 4 Culturing time(hour) FIGA INVENTOR ATTORNEY METHANOL-ASSIMILATING PROPAGATION OF MICROBIAL CELLS BACKGROUND OF THE INVENTION The present invention relates to a process for the propagation of microbial cells and is more specifically directed to a process for propagation of methanolassimilating microbial cells by aerobically culturing Pseudomonas methanolica in a medium containing methanol.

Propagation of microbial cells to recover fermentation products or to recover cell protein is well known. Moreover, propagation of microbial cells in a medium containing hydrocarbons as the assimilable carbon Size: 0.8 x (1.5 2.0) p. Shape: rod

Spore: none Motility:

Motile by means of single polar flagellum, of which electron-microscopic photo (magnification: 10,000 X) isshown in FIG. 4 of the accompanying drawings.

Colonies: circular; raised; entire; pale pink Pigment: pale pink; soluble in water; insoluble in organic solvents; non-diffusible b. Physiological properties 1. Optimum growth conditions: pH 7.0; temperature 30C; aerobic.

source is also known. The benefits obtained by using 2. Conditions for growth: pH 6.8 8.0; temperature hydrocarbons as a carbon source are also known par- 40C; aerobic ticularly with respect to the savings incurred in indus- 3. Gram-negative trial processes. However, many hydrocarbons have not 4. Non acid-fast been suitable for cultivation of microorganisms since 5. Methyl red test: negative the hydrocarbons could not be economically assimi- 20 6. Voges-Proskauer reaction: negative lated by any known microorganism. 7. lndole: not produced Heretofore, attempts have been made at culturing 8. Hydrogen sulfide: not produced microbial cells using methanol asthe assimilable car- 9. Ammonia: not produced bon source, but there has not, until now, been a satis- 10. Reduction of nitrates: positive factory method wherein there is an industrially feasible l l. Catalase: positive rate of propagation and yield of microbial cells. To this 12. Gelatin and casein: not liquefied V end, the present inventors have isolated microorganl3. Starch: not hydrolized isms which assimilate methanol and may be propagated l4. Citric acid: not utilized in a medium containing methanol as the carbon source 15. Milk: not coagulated in an industially feasible manner. 16. Litmus: not reduced; methylene blue and 2,6-

dichloro henol indo henol: reduc d SUMMARY or THE INVENTION P 17. Ammonium salts: utilized, urea. utilized The present invention provides a process for propac. Utilization of the following carbon sources: gating methanol-assimilating microbial cells belonging to a new species of microorganism. The new species is named P e dom nas methanol ca and ma be aerobi- Ammo: not uulwed s u 0 l y Y c. Utilization of the following carbon sources: cally cultured in a medium containing methanol resulting in rapid propagation of the cells. L Arabinose. not utilized The newly isolated microorganism 18 characterized 2. Xylose: by the following properties: a a. Growth state on each of the following media 5. Fructose: 6. Galactose: 1. Bou llon. no growth 7' mm: H 2. Bouillon agar. no growth Manon, I 3. Glucose bouillon agar: no growth :bsa cctg roae: re osc: 4. Gelatin. no growth Rafi-mm: not "mind 5. Peptone water: no growth 12. Sorbitol: 6. Litmus milk: no growth lnm'mli 14. 0| cerol: 7. Potato: no growth 15. S icin: 8. Methanol-containing synthetic medium (The com- F l y s position shown below): very excellent growth Composition of the medium 1'). Starch: 20. Cellulose: 21. Methanol: utilized Potassium dihydrogenphosphate 0.5g Ammonium monohydrogenphosphate 2.0g Methane 9' uhzed Potassium chloride 0.1g Magnesium sulfate 8 Isl-{pus slulfate -g l s The microbial characteristics of this new microorg w 1.0mm ganism in comparison with the well-known, methanol- PH 7.0 assimilable strains are given in the following Table 1.

TABLE 1 Newly isolated Paeudomonas .llethanomonas strain ATCC Strains Psrwdomonasmethanica A.\I1 methanooridans 21704" Slzv 0.8 x 2.0.. 1.0 x(1.54.0).... 0.3 x (1.54.0). (lmm slain Negative... \egative Negative Negative" Negative. l lagelln P 2. Polar Polar Polar. Requirement for growth fact 0)... None Not disclosed None. Pigment:

Solubility ..d0..... Color Red Red .do Pale pink (yellow).

TABLE l-(kvnlinlicd Newly isolated Pseudomonas .Uethanomonas strain ATCC strains Psceudomonasmethanica AM-t methanooridans 21704" Absorption wave length NOt disclosed... 520 550111;].

Presence of poly-B-hydroxybutyrate Fructose... Forinic acid Suecinic acid Malic acid Fuinoric acid Citric acid 1 Agar extract solution or pantothcnio acid. 2 Water-insoluble, organic solvontsolublo. 3 Not disclosed organic solvent-soluble.

4 Red, brown, yellow or colorless.

As is noted in Table l, the newly isolated microorganism is characteristic of assimilating only methanol as a conventional carbon source, which is different from other similar methanol-assimilating microorganisms. Moreover, as to the solubility of the produced pigment, which is generally an important factor in identification of this type of bacteria, the pigment of the newly isolated strain is soluble in water but not in organic solvents. Further, the pigment has maximum absorbances at 520 my. and 550 mp. which are different from those of the pigments produced by the known similar strains. Thus, regarding these taxonomical differences as important, the present inventors identified the said isolated microorganism as belonging to a new species different from the known similar species and named the new species Pseudomonas methanolica. This microorganism has been deposited with the American Type Culture Collection, Rockville, Md., and has been given accession number 21704.

Specific embodiments of the invention have been chosen for purposes of illustration and description in the accompanying drawings, forming a part of the specification wherein:

FIG. 1 is a graph illustrating the relation between culturing time and yield of microbial cells in a single vessel culturing of Pseudamonas methanolica ATCC 21704 in a medium containing methanol;

FIG. 2 is a schematic view of a double tank culturing apparatus;

FIG. 3 is a graph illustrating the relationship between culturing time and concentration of microbial cells in an alternative culturing process; and

FIG. 4 is an electron microscope photograph (10,000 x) of Pseudomonas methanolica ATCC 21704.

With reference to FIG. 1, Pseudomonas methanolica ATCC 21704 is aerobically cultured at 30C in a medium having the following composition Patassium dihydrogenphosphate 0. Ammonium monohydrogenphosphate 2. Potassium chloride 0 Magnesium sulfate 0.2g Ferrous sulfate 0.01 Methanol m Distilled water 1,000ml pH 7.0

Curve II depicts Pseudomonas mrthanolica ATCC 21704 which is cultured in a flask under the conditions of an inoculum ratio of 2%(V/V) and an initial methanol concentration of 1% (V/V), the strain continues a logarithmic propagation after an inducing period of about one hour and completely consumes the methanol in a short period of about 9 hours. The specific propagation rate (p. max.) of the logarithmic propagation period of this strain is 0.49 (hr), and accordingly, the average time for doubling is about 1.4 hours which is very short as compared with that of known strains. This is a characteristic feature of the strain. Moreover, the yield of dry cells to the added methanol showed a very high value of about 50 percent. It has also been found that when the strain is cultured at a 0.2% (V/V) methanol concentration as shown by Curve I, the induction period is shortened. It has also been found that even at a 0.0 1% (V/V) methanol concentration, the value of umax. is not lowered.

The present inventors have also found that in carrying out the present invention any of the following means is suitable to prevent volatile loss of methanol during the aeration-agitation culturing in a culturing tank:

a. carrying out culturing in a single tank while keeping the concentration of methanol stationarily below 0.2% (V/V) by successively supplying methanol, and

b. carrying out culturing using a compound apparatus consisting of main and secondary culturing tanks as shown in FIG. 2. In this embodiment, a medium containing comparatively high concentration of methanol such as l 2% (V/V) is placed in the main culturing tank (1) and a gas discharged therefrom containing volatilized methanol is led through a linking pipe (7) to a secondary culturing tank (2) (About one-fifth capacity of the main tank is sufficient) in which a medium containing a far lower concentration of methanol is placed and wherein the strain has previously been inoculated. The culturing according to the procedure (b) enables complete recovery of methanol as described below, and the total yield of cells from both tanks amounts to 54 percent to methanol. This is a considerable increase in yield compared with the procedure using a single tank. Furthermore, an attempt of accumulating microbial cells in high cencentration by feeding successively methanol to keep the methanol concentration at 1% (V/V) during the course of culturing according to procedure (b), has resulted in a remarkably high concentration of microbial cells within a short period of time as described below.

Continuous culturing is also appropriate for the present invention i.e., continuously applying culture medium to the culturing tank along with a partial discharge of the culture liquor.- the microbial cells are obtained at a rate of 0.42g/l/hr when the methanol concentration of the feed medium is adjusted to 0.2% (V/V) and the dilution ratio to 0.49. Continuous culturing may also be conducted while adjusting the methanol concentration of the medium to be supplied to 4.0% (V/V), and, as a result, 7.5g/l/hr of the cells are produced continuously. In the latter case, the methanol concentration of the medium in the culturing tank at equilibrium remains less than 0.2% (V/V) and, therefore, a secondary culturing tank for the recovery of volatilized methanol is practically unnecessary.

The propagated microbial cells are recovered by a conventional method such as centrifugation.

Practice of certain specific embodiments of the invention is illustrated by the following representative examples.

EXAMPLE 1 In this example, a culturing apparatus comprising a main culturing tank (1) and a secondary culturing tank (2), as shown in FIG. 2, is utilized. Culturing is carried out under the conditions below described, with aeration from a circular sparger (4) equipped at the .end of an aeration pipe (3) in the liquid bottom together with agititaion by an agitator (5) and baffles (6), while the culturing of the liquid in the secondary tank (2) is carried out under the conditions below described. A gas discharged from the main tank containing volatilized methanol is led to the secondary tank through a linking pipe (7) connecting both tanks. The pipe (7) terminates at the bottom part of the secondary tank as a circular sparger (8) whereby the volatilized methanol is introduced into the culture medium of the secondary tank.

A strain of Pseudomonas methanolica ATCC 21704 is inoculated into both tanks and the following culturing conditions are met:

Main tank Secondary tank Capacity l liters 2 liters Amount of medium liters 1 liter Revolutions 800 rpm 400 rpm Amount of aeration l vvm l vvm Culturing temperature 30C 30"C Culturing time 10 hrs 10 hrs initial methanol concentration I% (WV) 0 2% (WV) Concentration of the cells 3.6g]! 1.6

Com ositlon of the medium except methanol: Potassium dihydrogen-phosphate 0.5g Ammonium monohydrogenphosphate 2.0g Magnesium sulfate 0.2g Potassium chloride 0. lg Ferrous sulfate 0.0lg Distilled water l,000ml pH 7.0

As a result, the methanol recovery to the secondary culturing tank (2) is carried out completely, and total yield of the cells from these two tanks amounts to 54 percent to methanol. The cells are recovered by contrifugation.

EXAMPLE 2 In this example, a strain of Pseudomonas methanolica ATCC 21704 is inoculated into .main and secondary tanks and culturing is carried out with aerationagitation under the following conditions while successively supplying the tank with methanol at intervals of time marked by the arrows in FIG. 3. ln such manner the methanol concentration of the culture medium is maintained at 1.0% (V/V). Culturing conditions:

Composition of the medium Potassium dihydrogenphosphate 0.5g Ammonium monohydrogenphosphate 2.0g Potassium chloride 0.1 g Magnesium sulfate (heptahydrate 0.2g Ferrous sulfate (heptahydrate) 0.0l g Distilled water l,000ml pH 7.0 Culturing temperature C The increase in the cell concentration with the lapse of culturing time is shown in FIG. 3. As is shown in the figure, about 20g]! of the cells are accumulated after culturing for 20 hours and the yield amounts to about 50 percent to methanol. The accumulated cells are recovered by centrifugation.

EXAMPLE 3 In this example, a strain of Pseudomonas methanolica ATCC 21704 is inoculated into a 1 liter fermenter and continuous culturing is carried out under the following conditions while supplying the fermenter with a medium and partially discharging the culture liquor therefrom, continuously. The produced cells are recovered from the discharged culture liquor by centrifugation. Culturing conditions:

Composition of the medium Potassium dihydrogenphosphate 0.5g Ammonium monohydrogenphosphate 2.0g Potassium chloride 0.1g Magnesium sulfate 0.2g Ferrous sulfate 0.0l g Methanol 2ml Distilled water l,000ml P" 7.0

Maximum amount of cells produced;

0.83 fill 0.42 gll r Yield of cells: 52%

EXAMPLE 4 In this example, continuous culturing is carried out in the same manner as in Example 3 except with a methanol concentration of 4.0% (V/V) in the feed medium. The following results are obtained:

Stationaryconcentration of cells: 15.3 [ill Maximum amount of cells produced: 7.5 g]! r Yield of cells: 48%

An analysis of the composition of the cells (dry product) obtained through the continuous culturing in Example 4 shows that the product cells contain, in percent, the crude protein containing the following amino acids, which is a protein of good quality. Similar results are obtained from the product cells of Examples 1, 2

and 3.

Composition of amino acids in dry cells lysine 3.4 alanine 5.6 histidine l.3 valine 4.4 arginine 3.8 methionine 1.3 asparagine 9.4 isoleucine 39 threonme 4.1 leucine 5.9 serine 3.6 tyrosine 2.4 glutamic acid 10.0 phenylalanine 3.4 glycine 4.2 tryptophane 0.6 proline 5.1 cystine 0.3

carried out in a single tank fermenter and said process further includes the step of maintaining the concentra tion of methanol in said culture medium stationarily below 0.2% (WV).

4. the process of claim 1 wherein said culturing step includes the steps of culturing said strain in a main culturing tank containing a culture medium having a methanol concentration of from 1 to 2% (V/V), and venting volatilized methanol into a secondary culturing tank containing an inoculated culture medium having a methanol concentration less than said main tank to efiect complete recovery of methanol.

5. The process of claim 1 wherein said culturing step includes the steps of continuously supplying culture medium to a culturing tank and partially discharging culture liquor from said tank while maintaining the methanol concentration'in said culturing tank to less than 0.2% (V/V). 

2. The process of claim 1 wherein said strain of Pseudomonas methanolica is Pseudomonas methanolica ATCC
 21704. 3. The process of claim 1 wherein said culturing is carried out in a single tank fermenter and said process further includes the step of maintaining the concentration of methanol in said culture medium stationarily below 0.2% (V/V).
 4. the process of claim 1 wherein said culturing step includes the steps of culturing said strain in a main culturing tank containing a culture medium having a methanol concentration of from 1 to 2% (V/V), and venting volatilized methanol into a secondary culturing tank containing an inoculated culture medium having a methanol concentration less than said main tank to effect complete recovery of methanol.
 5. The process of claim 1 wherein said culturing step includes the steps of continuously supplying culture medium to a culturing tank and partially discharging culture liquor from said tank while maintaining the methanol concentration in said culturing tank to less than 0.2% (V/V). 